Synthetic guide rna

HT-29 cells were transformed by clustered regularly interspaced short palindromic repeats endonuclease-mediated mutation with Cas9 2NLS nuclease (Synthego) and a synthetic guide RNA (Synthego) for human cytochrome p450 family 27 subfamily B member 1 (CYP27B1) (5′-GUGGUACUCUCGGUAGCCUA-3′). Transformations were performed by using Lipofectamine 3000 (Invitrogen) with Cas9 2NLS nuclease and synthetic guide RNA in Opti-Mem medium (Gibco). Mutagenesis was verified by Western blot analysis of the expressed CYP27B1 protein, 1α-OHase.

For genome editing of donor hCD34⁺ cells, CAS9 ribonucleoprotein (RNP) complex was prepared by diluting 300 pmol of in vitro-transcribed (synthesized as described³⁶ ) or synthetic guide RNA (Synthego) and 500 pmol of purified CAS9 (University of California, Berkeley MacroLab) to a final volume of 20 µL of CAS9 buffer as previously described.^37,38 (link) Approximately 1 × 10⁶ hCD34⁺ cells isolated from adult donor granulocyte colony-stimulating factor-mobilized peripheral blood were nucleofected using ER100 setting of Lonza 4D-Nucleofector in 100 µL of Lonza P3 solution + supplement (Lonza, V4XP-3012). Seventy-two hours after nucleofection, a fraction of the nucleofected cells were collected to assess the initial allele frequency using next-generation sequencing (NGS). The single guide RNA (sgRNA) sequences used are listed in Table 1.